Poly-.beta.-hydroxyalkanoates (PHAs) are bacterial storage polymers that are synthesized by bacteria when carbon source levels are high and other nutritional necessities, such as nitrogen, phosphate, oxygen, or sulfur, are limited (Anderson, A. J., E. A. Dawes, Microbiol. Rev. 54:450-472, 1990; Steinbuchel, A., H. G. Schlegel, Mol. Microbiol. 5:535-542, 1991). The first PHAs that were identified consisted of saturated 3-hydroxy fatty acids, but more recently identified polymers have been shown to contain double bonds (Huijberts, G. N. et al., Appl. and Environ. Microbiol. 58:536-544, 1992; Lageveen, R. G. et al., Appl. and Environ. Microbiol. 54:2924-2932, 1988).
General formulas for PHA polymers include the following: ##STR1## where the R group can range from a single methyl group (to provide a 3-hydroxybutyrate subunit) up to about a 13-carbon chain (to provide 3-hydroxyhexadecanoate). In addition, the R group may simply be a hydrogen (to provide 3-hydroxypropionate). In an alternative form, the PHA can have a 4-hydroxybutyrate monomer unit ("4HB") of the following general formula: ##STR2##
PHAs that consist essentially of, or are homogeneous for, 4HB monomer units are known as poly(4-hydroxybutyrate). PHAs comprising both 3-hydroxybutyrate monomer units and 4-hydroxybutyrate monomer units are known as poly(3-hydroxybutyrate-co-4-hydroxybutyrate). PHAs comprising 4HB are recently discovered and have attracted considerable interest because of their superior qualities for certain plastic applications. For example, P(3HB-co-4HB) has increased flexibility in thermoplastic uses (Kunioka et al., Appl. Microbiol Biotechnol. 30:569-573, 1989; Doi, Y., et al., Macromolecules 23:26-31, 1990; Doi, Y., et al., Int. J. Biol. Macromol. 12:101-106, 1990; Saito and Doi, Int. J. Biol. Macromol. 16:99-104, 1994).
PHAs have been synthesized by the action of three enzymes: .beta.-ketothiolase ("3-ketothiolase"), acetoacetyl-CoA reductase ("reductase"), and poly-.beta.-hydroxyalkanoate synthase ("PHA synthase") (see, e.g., Oeding and Schlegel, Biochem. J. 134:239, 1973; Senior and Dawes, Biochem. J. 134:225, 1973). These enzymes are encoded by genes known as phaA, phaB and phaC, respectively, and are known as the "polyhydroxyalkanoate biosynthetic pathway" (or "PHA biosynthetic pathway"). For example, in the production of poly-3-hydroxybutyrate ("P(3HB)") in Ralstonia eutropha, 3-ketothiolase condenses two acetyl-CoA molecules to acetoacetyl-CoA reductase (Ralstonia eutropha was formerly known as Alcaligenes eutropha (Yabuuchi, E. et al., Microbiol. Immunol. 39:897-904, 1995)). Acetoacetyl-CoA reductase reduces this compound to 3-hydroxybutyryl-CoA. PHA synthase then polymerizes 3-hydroxybutyryl-CoA into PHB. Other PHAs, including medium chain and long chain PHAs, are produced under other conditions, which can include pathways comprising enzymes other than phaA and/or phaB.
P(3HB-co-4HB) has been produced from the above pathway by feeding expensive precursors such as 4-hydroxybutyrate, 1,4-butanediol or 4-butyrolactone to wild type or mutant strains of Ralstonia eutropha. Normally the molar levels of 4HB in the copolymer are relatively low, but .gamma.-butyrolactone has reportedly been fed to Alcaligenes latus to obtain P(3HB-co-4HB) levels that are approximately 60% of the cell dry weight and which contain 7-12 mol % 4-hydroxybutyrate (4HB) monomers (Soejima and Doi, International Symposium on Bacteriol Polyhydroxyalkanoates, Davos, Switz., 1996).
Steinbuchel et al., J. Env. Pol. Deg. 2, 67-74, 1994, have isolated A. eutrophus mutants that were able to accumulate a P(4HB) homopolyester. When these mutants were supplemented with the A. eutrophus PHA synthase gene, P(4HB) homopolyester was accumulated to levels of approximately 30% of the cell dry weight.
The field of PHA production has altered its focus in recent years to include plant produced PHAs (Eschenlauer, A. C. et al., Abstr. Annul. Meet. International Symposium on Bacterial Polyhydroxyalkanoates, Montreal, Canada, 85:66, 1994; Hahn, J. J., Abstr. Annu. Meet. International Symposium on Bacterial Polyhydroxyalkanoates, Montreal, Canada, 82:65; Landschulze, V. et al., Abstr. Annu. Meet. International Symposium on Bacterial PHA, Montreal, Canada, 86:66; Nawrath, C. et al., Abstr. Annu. Meet. International Symposium on Bacterial Polyhydroxyalkanoates, Montreal, Canada, 83, 1994; Nawrath, C., Proc. Natl. Acad. Sci. 91:12760-64, 1994; Poirier, Y., Adv. Mater. 5:30-36, 1993; Pool, R., Science 245:1187-1189, 1989; Poirier, Y., Sci. 256:520-523, 1992). Such technology includes the expression of the A. eutrophus pha synthesis genes in Arabidopsis thaliana (Poirier, Y., Sci. 256:520-523, 1992) with the formation of PHA reaching 14% of the dry weight of the leaves (Nawrath, C., et al., Proc. Nat. Acad. Sci. USA 91:12760-12764). PHA production in plants may be less expensive than PHA production in bacteria.
Further information with respect to the production of PHAs can be found in U.S. Pat. Nos. 5,334,520, 5,371,002, 5,512,456, and 5,569,595, and in PCT applications PCT/US93/05187, PCT/US94/03252, PCT/US95/01433, and PCT/US95/01471, as well as in Kidwell, J., Valentin, H. E., and Dennis, D., Appl. Environ. Microbiol. 61:1391-1398, 1995), Huisman, G. W., et al., Genetic Analysis Of Polyester Synthesis, p. 451-452, in E. A. Dawes (Herausg.), Novel Biodegradable Microbial Polymers, Kluwer Academic Publishers, Netherlands, 1990; Slater, S., et al., Appl. Microbiol. 58:1089-1094, 1992.
A disadvantage with present production systems for PHA comprising 4HB monomer units is that they are too expensive for practical application, for example, because of the high-cost carbon substrates that are precursors of 4HB monomer units found in PHA. Conversely, the less expensive P(3HB) is brittle, and thus not usable for many applications, while polymers comprising significant amounts of polyhydroxyoctanoate (and/or longer chain subunits) are too flexible for many plastic uses, and thus similarly unsuitable for many applications. PHA comprising 4HB monomer units would provide many useful plastic properties not found in other PHAs (Saito, Y. et al., Int. J. Biol. Macromol. 16:99-104, 1994).
Thus, there is a need in the art for methods and constructs capable of producing PHA comprising 4HB monomer units in large amounts in an efficient, economic manner. The present invention fulfills this need and provides other related advantages.